In order to understand the etiology of liver cancer, it is necessary to determine the mechanisms by which liver tumor promoting agents function. Though phenobarbital (PB) has been known for 20 years to be a potent liver tumor promoter, it molecular mechanisms of action are still not completely known. During the past grant period, we have discovered three separate steps in the mitogenic signaling pathways which are perturbed by PB. These include the impairment of two pathways involved in stimulating normal hepatocyte proliferation and the enhancement of a mito-inhibitory pathway. These findings support the postulate that the clonal expansion of initiated hepatocytes results from a growth advantage due to their refractoriness to the net anti-proliferative environment produced by PB. In this grant application, we have proposed experiments designed to determine the function of growth factors, growth factor receptors and tumor suppressor genes in liver tumor promotion. The primary objectives of this application are as follows: (1) We will determine whether the IGF-II/M6P receptor is involved in the hepatocyte activation of TGF-beta1. If we find that TGF-beta1 is activated subsequent to IGF-II/M6P receptor binding, it suggests that the overexpression of the IGF-II/M6P receptor in response to PB is critical in reducing normal hepatocyte's ability to replicate in response to mitogens. This postulate will be further investigated with the use of transgenic mice which contain the IGF-II/M6P receptor under the regulation of the metallothionein promoter. (2) Since TGF-beta1 is elevated in hepatocytes chronically exposed to PB, and TGF-beta1 prevents cell proliferation by inhibiting the phosphorylation of the Rb tumor suppressor gene, we have proposed to investigate the effect of PB on the regulation of the Rb gene and of another tumor suppressor gene highly expressed during liver regeneration, the p53 gene. (3) We will determine whether PB, in addition to reducing the capacity of normal hepatocytes to proliferate, also selectively enhances the proliferation of preneoplastic hepatocytes by either increasing the plasma concentration of Hepatocyte Growth Factor (HGF) or stimulating preneoplastic hepatocytes to directly produce HGF. (4) We will determine whether preneoplastic hepatocytes are refractory to PB-induced perturbations in the EGF receptor functions we previously have described for normal hepatocytes, and whether the decreased EGF receptor functions we previously have described for normal hepatocytes, and whether the decreased EGF binding on the surface of PB-exposed hepatocytes results from altered EGF receptor glycosylation. (5) We will investigate whether preneoplastic hepatocytes are refractory to the impairment in PKC activation observed in PB-exposed normal hepatocytes, and whether this impairment in the ability to activate PKC also compromises the function of PKC-dependent mitogenesis pathways (ie. alpha1-adrenergic co-mitogenic activities). In summary, we will investigate at the cellular, biochemical, and molecular levels the mechanisms by which PB promotes hepatocellular tumor formation.